Microsoft Word - 11351 NSB Layachi 2022.03.16.docx Received: 16 Sep 2022. Received in revised form: 28 Jan 2023. Accepted: 07 Mar 2023. Published online: 16 Mar 2023. From Volume 13, Issue 1, 2021, Notulae Scientia Biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. The journal will continue to appear quarterly, as before, with four annual numbers. SHSTSHSTSHSTSHST Horticulture and ForestryHorticulture and ForestryHorticulture and ForestryHorticulture and Forestry Society of TransylvaniaSociety of TransylvaniaSociety of TransylvaniaSociety of Transylvania Layachi R et al. (2023) Notulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia Biologicae Volume 15, Issue 1, Article number 11351 DOI:10.15835/nsb15111351 Research ArticleResearch ArticleResearch ArticleResearch Article.... NSBNSBNSBNSB Notulae Scientia Notulae Scientia Notulae Scientia Notulae Scientia BiologicaeBiologicaeBiologicaeBiologicae Assessment of analgesic properties of alcohol and aqueousAssessment of analgesic properties of alcohol and aqueousAssessment of analgesic properties of alcohol and aqueousAssessment of analgesic properties of alcohol and aqueous eeeextractsxtractsxtractsxtracts of of of of Opuntia Opuntia Opuntia Opuntia ficusficusficusficus----indicaindicaindicaindica flowersflowersflowersflowers Rajaa LAYACHI1a*, Aziz MOUHADDACH1b, Lamiae AMALLAH1, Mohammed BENAZIZ2, Fatima BOUAZZA1, Souad BENAICH3, Rachida HASSIKOU1, Souad SKALLI1 1Mohammed V University in Rabat, Faculty of Sciences, Plant and Microbial Biotechnologies, Biodiversity, and Environment Center, Morocco; r.layachi@um5r.ac.ma (*corresponding author); mouhaddach@gmail.com; l.amallah@hotmail.com; fatima.leader2020@gmail.com; r.hassikou@um5r.ac.ma; s.skalli@um5r.ac.ma 2Sultan Moulay Slimane University, Higher School of Technology, PB170, 54000, Khenifra, Morocco; m.benaziz@usms.ma 3Mohammed V University in Rabat, Faculty of Sciences, Physiology and Physiopathology Team, Morocco; s.benaich@um5r.ac.ma a,bThese authors contributed equally to the work AbstractAbstractAbstractAbstract The cactus (Opuntia ficus-indica (L.) Mill.) belongs to the Cactaceae family and it’s used in traditional folk medicine in treating a number of diseases and conditions. Due to the remarkable biological activity and to the bioactive (phytochemicals) compounds of O. ficus-indica, it becomes the aim of many research studies. The current study aimed to evaluate the analgesic activity of various solvent fractions (aqueous and ethanol) prepared from the O. ficus-indica flowers. The centrally analgesic potential was evaluated using tail flick latency in tail immersion and hot plate methods in mice. Morphine was used as a positive control at a dose of 3 mg kg- 1, s.c.. Intra-peritoneal administration of the aqueous extract of O. ficus-indica flowers at the highest dose did not produce any toxicity symptoms, thus the median lethal dose (LD50) was estimated to be greater than 2,500 mg kg-1. The results of the pain behavior evaluation according to the gender approach of mice showed that the pain tolerance threshold is high in males compared to females. We found that various plant extracts at doses of 300, 500, and 1,000 mg kg-1 i.p., displayed significant and dose-dependent protective effects (p < 0.01, p < 0.001, and p < 0.0001) as measured by increased latency time compared to vehicle control. The maximum anti- nociceptive effect was with the ethanol extract (71%) at 60 minutes at a dose of 1,000 mg kg-1, which was equivalent to the effect of morphine (70%). The results suggested that O. ficus-indica might possess significant analgesic effects, supporting the use of this plant in traditional medicine. Keywords:Keywords:Keywords:Keywords: centrally analgesic; flowers; Opuntia ficus-indica; morphine; traditional medicine IntroductionIntroductionIntroductionIntroduction Pain is one of the more complex pathological phenomena, this process involving the immune system, neurobiological processes and humoral systems (Smith et al., 1943). In the normal state, prostaglandins and https://www.notulaebiologicae.ro/index.php/nsb/index Layachi R et al. (2023). Not Sci Biol 15(1):11351 2 other molecules participate in the genesis and remediation of this anomaly (Sébastien, 2010). The current treatment for pain involves anti-inflammatory medications. Due to their undesirable side effects, which limit their uses, there is more interest in natural compounds extracted from medicinal plants. These plants have analgesic and antipyretic activity and could constitute an alternative therapy because of their accessibility and low toxicity (Queneau and Ostermann, 2004; Bussmann, 2011). The bibliographic survey reports that Opuntia ficus-indica (L.) Mill. (The plant name has been checked with http://www.theplantlist.org) is an important source of therapeutic agents, due to their pharmacological properties and their richness in bioactive molecules. O. ficus-indica has long been used in traditional medicine in various countries (Park et al., 2001; Mouhaddach et al., 2017). The data of the ethnobotanical survey carried out by Mouhaddach et al. (2017) showed that the various parts of the O. ficus-indica plant can be used for the treatment of various health conditions including gastric disorders, kidney pain, back pain, and leg pain. Cladodes (57%) were the most used part of the plant, followed by flowers (21%), seeds (11%) and roots (5%). Several parts of the plant have been used to relieve pain, especially cladodes and flowers. The O. ficus-indica flowers exert a wide range of pharmacological activities, including antioxidant, antimicrobial (Ammar et al., 2012), anti-ulcerogenic and healing activities (Ammar et al., 2015; Mouhaddach et al., 2017), as well as anti-inflammatory (Ahmed et al., 2005; Zou et al., 2005). O. ficus-indica has traditionally been used to reduce inflammation and previous studies have demonstrated its anti-inflammatory potential (Shirazinia et al., 2019). More recently, the anti-inflammatory and the analgesic effects of cladodes methanol extract have also been investigated by Siddiqui et al. (2016) and Mouhaddach et al. (2017). Within the available literature, few studies have focused on flowers compared to other parts of the plant (cladodes, fruits, and seeds). This study is the first report on analgesic effect of aqueous and alcoholic extracts of O. ficus-indica flowers via tail flick and hot plate tests. Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods Plant material O. ficus-indica flowers, spineless variety, were collected from the Rhamna region (Morocco) during the month of June 2015. Then they were dried for one week (45 °C) and ground into a coarse powder with the help of a suitable grinder. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. Preparation of aqueous and organic extracts The flower powder (50 g) mixed with distilled water (500 ml) was decocted at 100 °C for 15 minutes. Then the mixture was filtered, lyophilized (using a PHYWE chrisa lyophilizer) and stored at 4 °C. At the time of its administration the extracts were dissolved in 0.9% saline (NaCl). The organic flower extract was prepared via the Soxhlet method using ethanol as the extraction solvent. Animals Adult male and female Swiss mice (20-28 g, 2 months of age) obtained from the animal house, physiology and physiopathology laboratory, Faculty of Sciences, University Mohammed V, Rabat, Morocco, were used throughout these experiments. They were housed in cages at room temperature and 12:12 h light/dark cycle. The animals were provided with pelleted diet and tap water ad libitum. All experiments were carried out according to strict adherence to ethical guidelines (Zimmerman, 1983). Layachi R et al. (2023). Not Sci Biol 15(1):11351 3 Analgesic activity method Hot plate method Mice of both genders were selected and divided into five groups designated as group-I, group-II, group- III, group-IV and group-V, consisting of eight mice (4♀ and 4♂) in each group for control, positive control and test sample group respectively. Each group received a particular treatment, control group (0.9% NaCl solution, i.p.), positive control (Morphine 3 mg kg-1, subcutaneous, s.c.) and the test sample (ethanol and aqueous extracts of 300 mg kg-1, i.p., 500 mg kg-1, i.p., and 1,000 mg kg-1 i.p.). The animals were positioned on Eddy’s hot plate kept at a temperature of 53±0.2 °C. A cut off period of 15 seconds was observed to avoid damage to the paw (Franzotti et al., 2000). The latency (reaction) time was recorded when the mice licked their paws or jumped at 0, 30, 60, 90 and 120 minutes after intraperitoneal administration (i.p.) of the samples (Eddy and Leimback, 1953; Toma et al., 2003). Tail flick method Central analgesic activity was assessed according to the method described by Di Stasi et al. (1988) Mice in groups of eight (4♀ and 4♂) were held in position in a suitable restrainer with the tail extended. A 2-3 cm area of the tail was marked and immersed in a water bath thermo-statistically maintained at 53±0.2 °C. The withdrawal time of the tail from the hot water (in seconds) was noted at 0, 30, 60, 90, and 120 minutes as the reaction time or tail flick latency. The maximum cut-off time for immersion was 15 seconds to avoid tail injury. Control mice were treated with vehicle (0.9% NaCl solution, i.p.), while the reference group was given morphine subcutaneously (s.c.) (3 mg kg-1, s.c.). Flowers extracts at doses of 300, 500, and 1,000 mg kg-1 were administered by intra-peritoneal injection. Statistical analysis All values are expressed as mean ± S.E.M. (standard error of mean). Data were analyzed by one-way ANOVA (analysis of variance) followed by Tukey’s multiple comparison tests. A level of p < 0.05 was considered statistically significant. Results Results Results Results Acute toxicity of the aqueous extract The mice showed no change in general physical appearance and somatomotor skills during the observation period. No manifestations of tremors, convulsions, salivation, diarrhea, coma, or behaviors were observed. However, manifestations of sleep were observed in treated mice during the first 30 minutes after gavage. No other treatment-related changes or mortality were observed in mice over the 24-hour period following oral administration of a single dose of O. ficus-indica flower decoction at a dose of 2,000 mg kg-1. The median lethal dose (LD50) was therefore estimated to be greater than 2,500 mg kg-1. Analgesic effect Tail Flick Test Tables 1 and 2 represent the results obtained via the tail immersion test to test the analgesic effect of aqueous and organic extracts of O. ficus-indica flowers. The results showed that the extracts at doses of 300, 500 and 1,000 mg kg-1, i.p. induce significant analgesic activity, demonstrated by an increase in latency time in seconds (Table 1) compared to the control. The tail withdrawal latency time of control mice was 2.21±0.43 s. At 30 minutes after administration of the extracts, we found that the latency times of the mice of the batches receiving the two aqueous and ethanol Layachi R et al. (2023). Not Sci Biol 15(1):11351 4 extracts at doses of 300, 500 and 1,000 mg kg-1, i.p. as well as that of the mice of the batch receiving morphine at 3 mg kg-1, s.c., were significantly higher (p < 0.001) compared to the control group. The latency time of the batch that received the extract dosed at 1,000 mg kg-1, i.p., was significantly higher (p < 0.05) than that of the batch that received the extract at 500 mg kg-1, i.p. Similarly, the batch which received the extract at a dose of 500 mg kg-1, i.p., turns out to have a significantly higher latency time (p < 0.05) than that of the batch which received the extract at 300 mg kg-1, i.p.. As for the percentage of pain inhibition, it was noted that the percentage of pain inhibition after 60 minutes with morphine at a dose of 3 mg kg-1 was 70%. This value was reached at a dose of 1,000 mg kg-1 of the aqueous extract. Also, the pain inhibition percentage of the ethanol extract after 60 minutes was slightly higher at 71%. Table 1.Table 1.Table 1.Table 1. Analgesic effect of aqueous and organic extracts of O. ficus-indica flower, latency in tail flick test in mice Treatments Doses mg kg-1 Reaction time ± S.E.M. 0 min 30 min 60 min 90 min 120 min Vehicle 0.2 2.21 ± 0.43 2.56 ± 0.54 2.06±0.14 2.56±0.61 2.74±0.33 Morphine 3 2.78 ± 0.78 8.6 ± 0.95*** 9.38±1.55*** 9.03±1.23*** 6.29±0.89** Ethanol extract 300 3.21 ± 0.54 4.55 ± 0.11*a 6.93±0.69**c 4.53±0.39*c 3.77±0.34c 500 3.06 ± 0.43 7.04 ± 0.88**b 8.37±0.73***b 6.98±0.35**c 4.74±0.37b 1,000 2.32 ± 0.47 7.20 ± 0.87***c 8.12±0.97***a 7.10±0.85***c 4.75±0.54b Aqueous extract 300 2.04 ± 0.22 3.85 ± 0.12*c 5.39±0.15***c 4.90±0.21**c 3.93±0.21b 500 2.73 ± 0.59 5.63 ± 0.23**b 8.22±0.54***b 6.51±0.30**c 5.51±0.17b 1,000 2.71 ± 0.61 5.57 ± 0.15**b 8.65±0.27***b 6.57±0.18**c 5.77±0.16*b Results are presented as mean ± SEM (n=8), analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (*): p < 0.01; (**): p < 0.001; (***): p < 0.0001 for difference in change from vehicle control group, (a): p < 0.05; (b): p < 0.001; (c): p < 0.001 for difference in change from standard reference group. Table 2.Table 2.Table 2.Table 2. Pain inhibition percentage for different treatment types in the tail flick test Treatments Doses mg kg-1 Inhibition percentage (%) 30 min 60 min 90 min 120 min Morphine 3 68 70 69 56 Ethanol extract 300 29 53 29 14 500 56 63 56 35 1,000 68 71 67 51 Aqueous extract 300 47 62 58 48 500 51 68 58 50 1,000 68 70 69 56 Hot plate test Results from hot plate testing of latency time and inhibition percentage are presented in Tables 3 and 4. Morphine at dose of 3 mg kg-1, the aqueous and ethanol extract at doses of 300, 500 and 1,000 mg kg-1, showed a significant increase in the latency time. The latency time results at different doses showed a significant increase, hence a dose-dependent effect. The highest pain inhibition percentage (69%) for the aqueous extract was obtained by the highest dose 1,000 mg kg-1, while the percentage for ethanol extract was 65%. Layachi R et al. (2023). Not Sci Biol 15(1):11351 5 Table 3.Table 3.Table 3.Table 3. Analgesic effect of aqueous and ethanol extracts of O. ficus-indica flowers on latency via the hot plate test Treatments Doses mg kg-1 Reaction time ± S.E.M. 0 min 30 min 60 min 90 min 120 min Vehicle 0,2 2.48±0.27 2.42±0.63 2.32±0.23 2.53±0.25 2.23±0.72 Morphine 3 2.48±0.86 8.34±0.26*** 9.62±1.59*** 8.33±1.22*** 6.24±0.78** Aqueous extract 300 2.34±0.25 3.44±0.19c 5.03±0.22**c 4.50±0.13*c 3.60±0.22c 500 2.22±0.28 3.72±0.22c 6.32±0.27**b 4.97±0.23**c 3.41±0.15c 1,000 2.43±0.19 4.88±0.15**c 7.92±0.18***b 5.46±0.20**c 3.84±0.18c Ethanol extract 300 2.78±0.43 3.77±0.38c 6.50±1.16**c 4.64±0.38*c 3.63±0.28b 500 2.65±0.88 6.88±1.53**b 6.86±0.83**c 5.97±0.93**b 4.05±0.18b 1,000 2.66±0.85 5.81±0.71*c 7.54±0.43***b 6.18±0.62**c 4.25±0.35b Results are presented as mean ± SEM (n=8), analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (*): p < 0.01; (**): p < 0.001; (***): p < 0.0001 for difference in change from vehicle control group, (a): p < 0.05; (b): p < 0.001; (c): p < 0.001 for difference in change from standard reference group. Table 4.Table 4.Table 4.Table 4. Pain inhibition percentage for different treatment types in the tail flick test Treatments Doses (mg kg-1) Inhibition percentage (%) 30 min 60 min 90 min 120 min Morphine 3 70 74 70 60 Aqueous extract 300 32 53 48 35 500 40 65 55 35 1,000 50 69 55 37 Ethanol extract 300 26 57 40 23 500 61 61 56 34 1,000 54 65 57 37 Pain behavior assessment according to the gender of mice Pain inhibition results of aqueous extract of O. ficus-indica flowers obtained via experimental hot plate model in male and female mice are shown in Figures 1, 2 and 3. The pain behavior results in male and female mice according to the hot plate test showed a significant difference (p < 0.05; p < 0.01). Then, this difference was observed for all tested doses. Finally, these results show that the pain tolerance threshold is high in males compared to females (the graphs have been represented with reference to the tail flick test model results). Layachi R et al. (2023). Not Sci Biol 15(1):11351 6 Figure 1.Figure 1.Figure 1.Figure 1. The percentage of pain inhibition according to mice gender after administration of the aqueous extract at 300 mg kg-1 Figure 2.Figure 2.Figure 2.Figure 2. The percentage of pain inhibition according to mice gender after administration of the aqueous extract at 500 mg kg-1 Figure 3.Figure 3.Figure 3.Figure 3. The percentage of pain inhibition according to mice gender after administration of the aqueous extract at 1,000 mg kg-1 0 10 20 30 40 50 60 70 30 min 60 min 90 min 120 min P a in i n h ib it io n p e rc e n ta g e (% ) Reaction time (min) males females 0 10 20 30 40 50 60 70 80 30 min 60 min 90 min 120 min P a in i n h ib it io n p e rc e n ta g e (% ) Reaction time (min) males females 0 10 20 30 40 50 60 70 80 30 min 60 min 90 min 120 min P a in i n h ib it io n p e rc e n ta g e (% ) Reaction time (min) males females Layachi R et al. (2023). Not Sci Biol 15(1):11351 7 DiscussionDiscussionDiscussionDiscussion In the present study, we first examined the toxicity of the aqueous extract and also the anti-nociceptive potential of O. ficus-indica flower extracts (aqueous and ethanol) as a central analgesic using tail flick and hot plate test followed by the reaction to the painful behavior according to the gender of the mice. Firstly, oral administration of the highest dose of 2,000 mg kg-1 of O. ficus-indica flower extracts was found to produce no symptoms of acute toxicity. Although a slight sedation in the treated mice, no mouse mortality occurred during the 24 hours observation period. The extracts were found to be non-toxic at the highest dose (2,000 mg kg-1), thus, the LD50 was therefore estimated to be greater than 2,500 mg kg-1 according to the OCDE decision tree (OCDE, 2001). The highest dose dilutions were designated to assess analgesic activity, namely 300, 500 and 1,000 mg kg-1. The impact of gender on the response to an experimental nociceptive stimulus has been the topic of few studies. Thus, the results obtained within the present work have shown that the pain tolerance threshold is higher in males compared to females. Similarly, Mogil et al. (2000) according that female rats are more sensitive to thermal stimuli compared to males. This peculiarity probably results from the dynamic interaction of psychological and physiological factors. Concerning the analgesic impact study, the anti-nociceptive tests used in the current work were chosen to test the thermal cutaneous stimuli (Tail flick and hot plate tests). Sayyah et al. (2004) demonstrated that thermal stimuli are selectively inhibited by central analgesics however not peripheral analgesics in rats. We have used morphine in this study to test our hypothesis. The obtained results showed a significant difference compared to control for three doses tested, so the two extracts of O. ficus-indica flowers would have a central analgesic impact. Therefore, these results justify their significant protective effect and dose-dependent response to the thermo-painful stimulus. The analgesic effect proven by the tested extracts was approximately comparable to that of morphine. Indeed, at 3 mg kg-1 b.w., the pain inhibition of morphine was 70%. In the presence of flower extracts of O. ficus-indica at a dose of 1,000 mg kg-1, the inhibition percentage recorded is 70% for the aqueous extract and 71% for the ethanol extract, according to the tail flick test. In fact, the aqueous and ethanol extracts of O. ficus- indica flowers caused a reduction of painful behavior in mice. These results showed that O. ficus-indica flowers used for pain treatment (Mouhaddach et al., 2017) was justified. The obtained results by Chahdoura et al. (2017) showed that the aqueous extract of Opuntia microdasys flowers at a dose of 100 mg kg-1 produced a greater peripheral analgesic effect than that obtained with the positive control, lysine-aspirin (72% and 65.5% protection, respectively). These results are consistent with a few scientific studies reports that the plant has been used for their analgesic and anti-inflammatory actions. According to Park et al. (1998), fruit extract, freeze-dried cladodes and stem extracts had analgesic and anti-inflammatory actions. Considering the results prospected from ethnobotanical survey of O. ficus-indica, as well as those reported in the bibliographical review, we assumed that the richness of Opuntia in phenolic compounds, particularly in flavonoids, could produce a significant analgesic effect. Indeed, such a conclusion is supported by the phytochemical screening of O. ficus-indica which revealed the presence of bioactive molecules, mainly quercetin, nicotiflorin, narcissus, rutin, kaempherol, quercetin 3- O-methylether and 2,3-dihydrokaempferol (Lee et al., 2003; Guevara-Figueroa et al., 2010; Ammar et al., 2017; Abbas et al., 2022). Kuti (2004) reported that quercetin was the dominant flavonol in all analyzed species belonging to four different cactus species, namely O. ficus-indica, O. lindheimeri, O. strepthacantha, and O. stricta, and that this molecule has a remarkable analgesic effect. More recently, Ammar et al. (2017) reported that quinic acid was the predominant phenolic acid in the alcohol extract of Opuntia flowers, while quercetin-3-O-rutinoside was the major flavonoid identified. It was followed by quercetin-3-O-galactoside, quercetin-3-O-rhamonoside and isorhamnetin-3-O-glucoside. Also, the same study reports that this molecular combination produces a protective and anti-inflammatory effect Layachi R et al. (2023). Not Sci Biol 15(1):11351 8 according to the model of rat paw edema induced by carrageenan. In addition, fruit phytosterols have been identified as the main active anti-inflammatory component (Park et al., 2001; Izuegbuna et al., 2019). Therefore, the freeze-dried aqueous extract (100-400 mg kg-1, i.p.) of Opuntia dillenii fruits in hot plate assays in mice and rats possessed notable and dose-dependent analgesic activity (Loro et al., 1999). Furthermore, the obtained results in phenolic analysis are in agreement with previous studies reporting the presence of phenolic acids and flavonoids in the methanolic extract of O. ficus-indica flowers (Yeddes et al., 2014), as well as in the methanol extract of O. microdasys flowers (Chahdoura et al., 2014). Quinic acid was the major phenolic acid identified in this study, which accounted for 12.4% of the total identified phenolic compounds. This compound is also present in Moroccan O. ficus-indica with 3.65% of the total identified phenolic compounds (Benayad et al., 2014; Zeghbib et al., 2022). O. ficus-indica flowers extract was dominated by flavonol glycoside and the flavonol 3-O-glycosides (quercetin, kaempferol, and isorhamnetin) (De Leo et al., 2010; Kolniak-Ostek et al., 2020). These bioactive molecules also showed that certain compounds possessed analgesic activity. Also, flavonoids (kaempherol and isorhamnetin glycosides) are known to inhibit the prostaglandin synthetase enzyme, specifically endoperoxidase (Ramaswamy et al., 1985; Benattia et al., 219). Bentley (1983) and Negus et al. (2006) proved that the pain onset mechanism can be attributed to the release of substances in the intraperitoneal fluid such as prostaglandins (PGE2, PGF2 α), serotonin, histamine, bradykinin which will stimulate nociceptive receptors located in the peritoneum. In addition, the chemical composition of Opuntia flowers was characterized by the presence of flavonoids, saponins, tannins, and carbohydrates, contents (Ennouri et al., 2014). Thus, flavonoids are 5-lypo-oxygenase inhibitors, which allow inhibition of the inflammatory mediator’s effect (Ferreira, 2002). As a result, the presence of flavonoids in the O. ficus-indica flowers extract might contribute to the analgesic effect. Finally, we confirm that O. ficus-indica flowers possess a central analgesic activity, justifying their use in traditional medicine (Mouhaddach et al., 2017). This effect can be explained by the presence of phenolic compounds and flavonoids inhibiting the pain process. Other more advanced experimental models have made it possible to determine both the analgesic and anti-inflammatory activities of the aqueous extract obtained from Opuntia microdasys flowers. These interesting results are used in native anti-cancer regimens and suggest that Opuntia microdasys flowers could be explored for their pharmacological and antimutagenic profiles with potential utility of this plant within an herbal medicine or as a nutritional supplement. Furthermore, a more thorough analysis would allow us to discern the precise doses required for both anti-inflammatory and analgesic activities and, on the other hand, to quantify this activity by precisely establishing the action mechanism of mediating molecules in this process. Subsequently, it would be wise to seek a stable galenic formulation that can serve as a basis for this activity to enable design of a medicine to treat pain. ConclusionsConclusionsConclusionsConclusions In the present study we demonstrated the analgesic property of the O. ficus-indica flowers extracts, using conventional pharmacological models. These findings support its usage within the traditional pharmacopoeia. These properties are most likely related to flavonoids and phenolic compounds highlighted in the bibliography. Intra-peritoneal administration of O. ficus-indica flowers aqueous extract did not show any toxicity symptoms at the highest dose, thus the LD50 was estimated to be greater than 2,500 mg kg-1. The pain behavior results according to the gender approach of mice showed that the pain tolerance threshold is high in males compared to females. Further experiments using purified extracts are needed to accurately identify the active compounds responsible for this analgesic effect and to understand their action mode. Layachi R et al. (2023). Not Sci Biol 15(1):11351 9 Authors’ CoAuthors’ CoAuthors’ CoAuthors’ Contributionsntributionsntributionsntributions MA: Writing-Original Draft, Sampling and analysis; LR: Writing-Original Draft, Methodology; AL: Investigation, Resources; BM: Statistical analysis, Investigation; BF: Validation, Investigation; BS: Formal analysis, Investigation; HR: Supervision, Investigation, Resources; SS: Methodology, Validation. All authors read and approved the final manuscript. Ethical approvalEthical approvalEthical approvalEthical approval (for researches involving animals or humans) Not applicable. AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements This research received no specific grant from any funding agency in the public, commercial, or not-for- profit sectors. Conflict of InterestsConflict of InterestsConflict of InterestsConflict of Interests The authors declare that there are no conflicts of interest related to this article. 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